Investigating genomic diversity of Staphylococcus aureus associated with pediatric atopic dermatitis in South Africa

Importance Staphylococcus aureus frequently colonizes the skin and nose of patients with atopic dermatitis (AD), a disease associated with skin barrier dysfunction and chronic cutaneous inflammation. Published genomic studies on AD-associated S. aureus in pediatric populations in sub-Saharan Africa are limited. Objectives To investigate the phenotypic and genomic diversity of S. aureus in children with and without AD during early childhood. Data, setting and participants A cross-sectional study of 220 children (aged 9–38 months) with AD (cases) and without AD (controls) from Cape Town and Umtata, South Africa. Main outcomes and measures S. aureus phenotypic and genomic diversity were investigated using whole-genome sequencing, antibiotic susceptibility testing and biofilm microtiter assay. Results Of the 124 S. aureus isolates recovered from 220 children, 96 isolates (79 cases and 17 controls) with high-quality sequences were analyzed. Isolates from cases showed greater phenotypic resistance to gentamicin (10%), rifampicin (4%), chloramphenicol (4%), and exhibited multidrug resistance (9%) than in controls. Furthermore, the isolates from cases formed stronger biofilms than those from controls (76% vs. 35%, p = 0.001), but showed no dominance of any virulence factor gene or mobile genetic elements. There was no significant difference in the distribution of immune evasion cluster types between cases and controls. However, IEC type G was identified only among cases. Conclusion and relevance AD-associated S. aureus has phenotypic and genetic features that are important for successful pathogenic colonization and survival. Further studies are needed to assess the pathological implications of colonization of various S. aureus lineages in vivo to elucidate their pathological contribution to AD pathogenesis and pathophysiology.


Introduction
Pathological skin and nasal colonization with Staphylococcus aureus is common in patients with atopic dermatitis (AD)-a chronic or recurrent inflammatory skin disease which affects about 20% of children globally (Gilaberte et al., 2020).Due to the frequent association of S. aureus infections with AD, antibiotics are routinely used in the management of AD.However, the effectiveness of antibiotics in AD is limited by the rapid recolonisation with S. aureus following treatment cessation (di Domenico et al., 2019a).The excessive use of antibiotics may lead to significant changes in the skin microbial community and emergence of antibiotic-resistant isolates, which are associated with severe forms of AD (Jung et al., 2015;Shi et al., 2016).S. aureus contributes to AD pathology through virulence factors and biofilms, which are commonly detected in skin lesions of children with AD (Allen et al., 2014;Sonesson et al., 2017;di Domenico et al., 2018).Further, adhesins, toxins, proteases and significant changes in antigens enable the bacterium to adhere to host tissues, leading to impaired skin barrier function and inflammation (di Domenico et al., 2019b).Previous studies have shown that patients that are colonized with S. aureus strains which form strong biofilms, experience severe forms of the disease (Ogonowska et al., 2020;Gonzalez et al., 2021).
Some genes associated with antibiotic resistance, biofilm formation, and virulence in S. aureus are carried on mobile genetic elements (MGEs), such as plasmids, bacteriophages (prophages) and staphylococcal pathogenicity islands (SaPIs) (Malachowa and DeLeo, 2010).These are central to the adaptation of S. aureus in different environments (Malachowa and DeLeo, 2010).(Harkins et al., 2018) Moreover, lineagespecificity has been observed among MGEs (McCarthy et al., 2012;McCarthy and Lindsay, 2012).Recent data suggest that AD is associated with specific S. aureus lineages (Harkins et al., 2018), with clonal complex (CC) 1 being positively associated with AD in European children, while CC30 being negatively associated with AD (Rojo et al., 2014;Harkins et al., 2018).These differences in S. aureus lineages appear to be accompanied by differences in the carriage of virulence factors and adaptability to AD (Hwang et al., 2021).
It is therefore important to identify genomic markers of S. aureus that are associated with AD pathology in children with AD from their non-AD counterparts.Moreover, there is limited data on S. aureus strain biology from sub-Saharan Africa.We therefore investigated the phenotypic and genomic factors important for S. aureus adaptation on the skin and in the anterior nares of children with AD compared with healthy pediatric participants.

Collection of Staphylococcus aureus isolates
We used the S. aureus isolates collected from our previously published work (Ndhlovu et al., 2021).Briefly, samples were collected using sterile nylon-tipped flocked swabs (Cat.no.516C; Copan Italia, Brescia, Italy) from the lesional and non-lesional skin and anterior nares of children (n = 220) with and without AD from Umtata and Cape Town, South Africa (HREC/REF: 451/2014).The swabs were placed into skim milk-tryptone-glucose-glycerol (STGG) and transported to the laboratory within 2 h of collection at 4°C and stored in the freezers (−80°C) for further analysis.A total of 124 S. aureus isolates were recovered after the swabs were inoculated onto Mannitol Salt Agar (MSA) (National Health Laboratory Services [NHLS], Green Point Media Laboratory Cape Town, South Africa), and incubated at 37°C for 48 h in aerobic conditions.The S. aureus colonies were confirmed by testing for mannitol fermentation and DNase production (Kateete et al., 2010).Detailed information on the isolate collection can be found in Ndhlovu et al. (2021).

Staphylococcus aureus isolates
Antibiotic susceptibility testing (AST) was conducted using the Kirby-Bauer disk diffusion method as previously described and resistance to cefoxitin used as a surrogate for methicillin resistance (Wang et al., 2005).S. aureus ATCC 25923 was used as the quality control.The D-test was used to distinguish constitutive and inducible resistance to the macrolide-lincosamide-streptogramin B (MLS B ) group of antibiotics (Yao et al., 2019).The AST was interpreted according to 2021 EUCAST guidelines and breakpoints.Isolates that were resistant to at least three classes of antibiotics were classified as multidrug-resistant (MDR) (Magiorakos et al., 2012).

DNA extraction, library preparation, and whole-genome sequencing
Total DNA extraction was performed using a heat lysis method as described previously in Ndhlovu et al (Ndhlovu et al., 2021), and quantified using the Biotium Accuclear Ultra-high sensitivity dsDNA Quantitative kit.Samples were cherry-picked to 200 ng/120 μL using the Tecan liquid handling platform and sheared to 450 bp using a Covaris LE220 instrument.
Post sheared samples (in 96 well plates) were purified using SPRISelect beads and rearrayed into a in 384 plate on the Hamilton STAR.Library construction (ER, A-tailing, and ligation) was performed using the 'NEB Ultra II custom kit' on an Agilent Bravo WS automation system.PCR setup was performed using KapaHiFi Hot start mix and 384 well UDI tag barcodes on the Agilent Bravo WS automation system.PCR conditions were as follows: initial incubation at 95°C for 5 min followed by five cycles of 98°C for 30 s, 65°C for 30 s and 72°C for 1 min, and a final incubation step at 72°C for 10 min.Post PCR plates were pooled using equal volumes and purified using Agencourt AMPure XP SPRI beads on the Hamilton STAR.Libraries were quantified using an Agilent Bioanalyser, normalized to 2.8 nM, and prepared for sequencing on a NovaSeq (Page et al., 2016).

Phylogenetic tree construction and annotation
Single nucleotide polymorphism based phylogenetic analysis was conducted using the Reference sequence Alignment based Phylogeny builder (REALPHY) v1.13 (Bertels et al., 2014) with default settings.Staphylococcus aureus NCTC 8325 was used as a reference.The generated phylogenetic trees were visualised and annotated using the iTOL (Interactive Tree of Life) tool v6 (Letunic and Bork, 2021).

Discussion
This study shows that S. aureus isolates associated with AD tended to form stronger biofilms and carried genetic features that are linked  to antibiotic resistance and virulence with potential clinical relevance to AD pathology.The low prevalence of antibiotic resistance in our study is consistent with previous reports from Irish (Harkins et al., 2018), Polish (Wrobel et al., 2018), andItalian (di Domenico et al., 2018) pediatric patients with AD.The prevalence of MRSA strains was also low in both cases (8%) and controls (6%), as previously demonstrated (Harkins et al., 2018).Moreover, MDR S. aureus isolates were only detected among cases, suggesting frequent use of antibiotics or exposure to resistant strains, possibly from a hospital environment.Considering the low prevalence of AD in our setting, the proportion of MDR strains among children with AD may not reflect the true burden.Larger studies may be needed to further explore the impact of S. aureus antibiotic resistance on AD pathology.
S. aureus produces a variety of toxins, cytotoxins, proteases, antigens and adhesins that are key to its successful colonization, pathogenicity, and survival (di Domenico et al., 2019b).Epidemiological studies have shown that AD-associated S. aureus carry more virulence genes than controls (Na et al., 2012;Rojo et al., 2014;Cavalcante et al., 2015;Wang et al., 2016), which is also linked to increased disease severity (Rojo et al., 2014).Presently, the cases did not exhibit a predominance of virulence factor genes.Although no significant virulence factor genes were observed, it is likely that the disease environment could trigger more expression of the virulence factors resulting in disease pathology (Poh et al., 2022).
Biofilm formation is associated with increased disease severity (Pascolini et al., 2011;Allen et al., 2014;di Domenico et al., 2018).Particularly strong biofilms, protect S. aureus from (i) environmental factors (including antibiotics), (ii) the bactericidal effects of host antimicrobial peptides (AMPs) and, (iii) host immune responses (di Domenico et al., 2019a).In this regard, strong biofilm producers were more common among cases similar to previous studies (Allen et al., 2014;Sonesson et al., 2017;di Domenico et al., 2018).This suggests that AD-associated S. aureus isolates were better able to form biofilms and adapted for persistent colonization.These findings support the importance of strong biofilm formation in driving persistent colonization in children with AD.Also, persistent colonization with S. aureus in AD may be detrimental to the child as this provides a continuous source of virulence factors that will further perpetuate the clinical manifestations of AD and contribution to disease flares.As such, treatment strategies in AD need to have anti-biofilm capabilities to effectively reducing S. aureus colonization and mitigating its harmful effects.
Immune evasion by S. aureus is mediated by the immune evasion cluster (IEC), which modulates innate immune responses in humans (Verkaik et al., 2011).IEC includes SCIN, and a varying combination of SAK, CHIPS and staphylococcal enterotoxins, SEA or SEP, across different IEC types (van Wamel et al., 2006).We detected IEC genes in the S. aureus control group and most (92%) of the cases.Further, cases and controls did not differ substantially in the distribution of IEC types.Similar to previous reports (van Wamel et al., 2006;Verkaik et al., 2011;Silva et al., 2020), IEC type B, were independent of disease status, whereas the type G isolates were only identified among cases (Benito et al., 2016).Although the specific functions of the IEC genes and their contribution to AD are addressed in the literature (Rojo et al., 2014;Jian et al., 2021), the clinical relevance of different IEC types in AD is lacking and warrants further study.
Insertion sequences (IS) are small MGEs and are generally between 700-2,500 bp, affecting the genomic plasticity, diversity, and pathogenic potential of S. aureus (Kuroda et al., 2001).We found that ISCsp1 and ISSau6 were more common among controls.While there was no IS element more common in cases than controls, we observed that ISSau3, IS1181, ISSep3 were the three most frequently detected IS elements in cases.ISSau3 has been shown to increase resistance to beta-lactam antibiotics (Wang et al., 2021), the roles of IS1181 and ISSep3 are poorly studied in literature, especially in disease context.
There is increasing evidence that specific S. aureus lineages are adapted for induction of disease activity and survival in AD patients (Yeung et al., 2011;Byrd et al., 2017;Fleury et al., 2017;Harkins et al., 2018).In this study, we noted no significant difference in the clonality of S. aureus isolates from cases and controls.However, CC8 isolates predominated among cases, while CC121 isolates were the most common among controls.The specific features or mechanisms that favor the predominance of certain lineages in AD remain unknown (Geoghegan et al., 2018).However, it has been suggested that in AD, differences in the virulence potential across lineages are associated with the proliferation of some lineages and other lineages are associated with asymptomatic colonization in controls (Harkins et al., 2018).
Limitations to this study include the small sample size and our investigation focused on the recovery of a single colony which does not provide a comprehensive understanding of the clonality of S. aureus colonization in AD (Byrd et al., 2017).Lastly, we noted a higher prevalence of incomplete and questionable prophages compared to intact prophages.This is likely because intact prophages are usually under strong selection or genetic degradation for rapid deletion from bacterial genomes (Lawrence et al., 2001).However, since genomes are divided into contigs, some prophage sequences may have been split into different contigs and therefore misidentified as incomplete or questionable prophages by PHASTER (Marques et al., 2021).Moreover, the PHASTER tool also relies on previously annotated prophage sequences and, therefore, cannot identify novel prophage sequences.Subsequent studies will be focused on exploring the overall genomic factors beyond a specific set of genes, using tools like PRAWNS (Javkar et al., 2023).

Conclusion
This study provides an understanding of the genomic differences of S. aureus in early childhood AD, in a previously unstudied population in South Africa.Although we did not observe a clear phylogenetic and clonal differentiation of S. aureus based on AD disease, specific phenotypic and genetic signatures distinguished AD isolates from controls.Specifically, AD-associated isolates exhibited phenotypic features related to biofilm formation, and genomic features associated with DNA damage repair and toxins which are important for the persistent colonization and propagation of AD disease.
Table3shows the distribution of IS elements in cases and controls.ISSau3 was detected in all case and control isolates.

TABLE 1
The distribution of plasmids, present in at least 10 genomes, in cases and controls.

TABLE 2
The distribution of prophages, present in at least 10 genomes, in cases and controls.The information of the associated genes was obtained from the NCBI database.

TABLE 3
The distribution of IS elements, present in at least 10 genomes, in cases and controls.